Project Summary Testicular germ cell tumors (TGCTs) are the most common cancer in children and young men. TGCTs result from anomalies in the development of primordial germ cells (PGC), the embryonic precursors of gametes. Few factors involved in neoplastic transformation of PGCs have been identified in humans because the genetic component of TGCTs is complex and tumors initiate during embryogenesis. In 129/Sv mice, the only inbred strain of mice with an appreciable frequency of spontaneous TGCTs, tumors initiate around embryonic day 13.5 (E13.5). During this same developmental period, local retinoic acid (RA) levels in the developing gonad influences germ cell commitment to meiosis or mitotic arrest (the mitotic:meiotic switch). In female embryonic gonads, RA induces expression of Stra8, which functions in differentiation events required for germ cell entry into meiosis. In male embryonic gonads, RA is degraded, Stra8 expression is not induced, and gonocytes, the precursors of adult male germ cells, remain quiescent until after birth. The role of aberrant RA signaling and Stra8 expression in the premature differentiation of male embryonic gonocytes and the establishment of a tumor stem cell population in TGCT susceptible mice will be investigated. A combination of of in vitro and in vivo teratoma formation assays, expression assays, loss-of-function mutations, and pluripotency and tumorigenicity assays will be used to characterize three Specific Aims. Aim 1. Does aberrant retinoic acid signaling contribute to TGCT susceptibility and how are embryonic gonocytes exposed to RA? Aim 2. Does Stra8 function in embryonic gonocyte differentiation and TGCT susceptibility? Aim 3. Does a sub-population of embryonic gonocytes function as TGCT stem cells? These tests will reveal unique aspects of germ cell biology and TGCT tumorigenesis and may provide new targets for diagnosis and treatment of human TGCTs.